In Vitro Inhibition of Several Phytopathogenic Fungi from Avocado by Soluble Potassium Silicate T F Bekker1, C Kaiser1 and N Labuschagne2

Home , Lasiodiplodia theobromae, Uncinula necator

In vitro inhibition of several phytopathogenic fungi from avocado by soluble potassium silicate T F Bekker1, C Kaiser1 and N Labuschagne2

1Department of Plant Production and Soil Science 2Department of Microbiology and Plant Pathology University of Pretoria, Pretoria 0002, South Africa

ABSTRACT Silicon is a bioactive element only recently implicated as having fungicidal properties. The present study examined water soluble liquid potassium silicate for activity against several types of avocado phytopathogenic fungi. In vitro dose-responses towards soluble potassium silicate (20.7% silicon dioxide) were determined for Phytophthora cinnamomi, Phomopsis perniciosa, Pestalotiopsis maculans, Lasiodiplodia theobromae, Glomerella cingulata, Natrassia sp., and Collectotrichum gloeosporioides. Inhibition of mycelial growth was dose-dependant with 100% inhibition observed at 80 ml (pH 11.7) and 40 ml (pH 11.5) soluble potassium silicate (20.7% silicon dioxide) per litre of agar, for all fungi tested in two of the replicate experiments with the exception of Natrassia sp., G. cingulata and C. gloeosporioides at 40 ml in one replication. For both replicate experiments, Phytophthora cinnamomi, Phomopsis perniciosa, Pestalotiopsis maculans, Lasiodiplodia theobromae, Glomerella cingulata, Natrassia sp., and Collectotrichum gloeosporioides were only partially inhibited at 5, 10 and 20 ml soluble potassium silicate per litre of agar. Percentage inhibition was, however, positively correlated with soluble potassium silicate concentrations. Soluble potassium silicate raised the pH of the agar from 5.6 to between 10.3 and 11.7 at concentrations of 5 and 80 ml soluble potassium silicate per litre of agar respectively. The effect of pH on fungal growth does not follow a clear trend for all fungi tested. In the absence of soluble potassium silicate, high pH caused only partial inhibition of mycelial growth. Soluble potassium silicate therefore suppresses fungal growth effectively in vitro and this is largely a fungicidal effect.

INTRODUCTION of soluble potassium silicate against phytopathogenic fungi af- It has long been known that silicon reduces the incidence of fecting avocado. fungal diseases in various pathosystems (Fauteux, Rémus-Borel, Numerous avocado diseases are caused by pathogenic fun- Menzies & Bélanger, 2005). Numerous studies have shown in- gi present in the soil or air within avocado orchards. In many creased resistance to fungal plant diseases as a response to sili- instances the world has become reliant on chemical control of con applications. These include increased resistance to powdery these diseases, with the fear of fungicide resistance ever in- mildew (Uncinula necator) in grapes (Bowen, Menzies & Ehret, creasing (Hardy, Barrett & Shearer, 2001). 1992), powdery mildew (Sphaerotheca fuliginea) in cucumbers Development of fungicide resistant strains of phytopathogenic (Adatia & Besford, 1986; Belanger, Bowen, Ehret, & Menzies, fungi has been amply demonstrated. It is therefore imperative 1995), powdery mildew (Erysiphe cichoracearum) in muskmel- that alternative control measures should be investigated.The ons (Menzies, Bowen & Ehret, 1992) as well as Cladosporium current study was therefore initiated to assess the in vitro effect spp. and Pythium spp. in cucumbers (Chérif, Asselin & Belanger, of soluble potassium silicate on Phytophthora cinnamomi and six 1994). It was proposed that amorphous silica deposition in the other pathogenic fungi affecting avocado. leaf apoplast prevent penetration of fungal hyphae (Carver, Zey- en & Ahlstrand, 1987; Kunoh & Ishizaki, 1975). Although this in MATERIALS AND METHODS part explains the prophylactic properties of silicon, monomeric Fungi tested silicon is also implicated in activation of several plant natural Isolates of seven fungal pathogens of avocado maintained on defences (Fauteux et al., 2005), such as ﬂ avanoids phytoalexin potato dextrose agar (PDA) were obtained from the Mycological mediated response to silicon applications (Fawe, Abou-Zaid, herbarium of the Plant Protection Research Institute, Agricultural Menzies & Belanger, 1988) and prophylactic properties of silicon Research Council (ARC), Pretoria, and Westfalia Technological in plant defence reactions to fungal attack (Chérif et al., 1994). Table 1. Fungal pathogens of avocado tested for their in vitro sensitivity to soluble potassium Bekker, Kaiser, Van der silicate (20.7% silicon dioxide). Merwe & Labuschagne (2006) demonstrated for the ﬁ rst time that soluble potassium silicate has direct in vitro fungitoxic activity against a wide range of fungi. In the current study we are investigat- ing the fungitoxic activity

64 SOUTH AFRICAN AVOCADO GROWERS’ ASSOCIATION YEARBOOK 29, 2006 Services (WTS), Tzaneen, South Africa. Fungi were selected on silicate added) served as a control. the basis of availability of cultures as well as their importance as A 5 mm diam. mycelial disc taken from a 7 d old fungal culture avocado pathogens (Table 1). on PDA was transferred to the centre of a Petri dish containing soluble potassium silicate amended PDA at the various concen- Agar preparation trations, as well as unamended (control) plates respectively. Soluble potassium silicate solution was sterilized by passing This was done for each fungus. Ten replicates were included through a 0.45 μm Millipore fi lter before adding it to autoclaved for each treatment, with the entire experiment replicated twice. potato dextrose agar (PDA) to give fi nal concentrations of 5, 10, Plates were incubated at 25˚C in the dark and colony diameters 20, 40 and 80 ml soluble potassium silicate (20.7% silicon diox- were measured every second day for 8 consecutive days. ide) per litre of PDA. Soluble potassium silicate (20.7% silicon dioxide) has a pH of Percentage inhibition was calculated according to the formula: 12.7, which markedly increase the pH of PDA. A pilot study was thus conducted to determine the effect of various concentrations Percentage inhibition = (C-T) x 100 of soluble potassium silicate on the pH of PDA. It was found that C unameliorated PDA has a pH of 5.6. Upon addition of 5, 10, 20, Where C = colony diameter (mm) of the control 40 and 80 ml of soluble potassium silicate, the pH of the PDA is T = colony diameter (mm) of the test plate raised to 10.3, 10.7, 11.2, 11.5 and 11.7 respectively. This data was analysed with Genstat 4.23 DE (Table 2, 3 and The pH of PDA plates in the current study were therefore ad- 4). justed to pH 10.3, 10.7, 11.2, 11.5 and 11.7 using soluble potassium hydroxide. A non-ameliorated PDA (i.e. with no potassium RESULTS Soluble potassium silicate (20.7% silicon dioxide) completely Table 2. Mycelial growth of Phytophthora cinnamomi suppressed mycelial growth of all fungi (Table 3) at concentra- incubated for eight days at 25˚C on PDA. Agar was tions of 40 and 80 ml.l-1 PDA, with the exception of Natrassia sp., either amended with soluble potassium silicate G. cingulata and C. gloeosporioides (mean colony diam. of 5.4, (20.7% silicon dioxide) at various concentrations, or 11.5 and 13.1 mm respectively) in one experimental repetition. potassium hydroxide to increase pH value of the agar, A soluble potassium silicate concentration of 5-10 ml.ℓ-1 PDA after amendment with soluble potassium hydroxide inhibited mycelial growth of P. cinnamomi (Figure 1; Table 2), mimicking the pH effect of soluble potassium silicate. but in contrast increased fungal growth of P. perniciosa (Figure 2). Suppression of mycelial growth at concentrations of 5 to 20 ml.ℓ-1 PDA varied between all fungi tested and an effective sup- pressive concentration are to be determined for each fungus. Although PDA amended with soluble potassium hydroxide (pH range 10.3 to 11.7) did inhibit mycelial growth of most fungi to some degree (Table 3), the effect of added soluble potassium silicate over and above the pH effect is apparent. The effect of pH on mycelial growth as exemplifi ed by soluble potassium hydroxide amended PDA plates seems to be species-specifi c, and is not correlated to the inhibition due to soluble potassium silicate presence.

DISCUSSION The results of this study clearly indicate that soluble potassium silicate (20.7% silicon dioxide) has fungicidal activity in vitro. w pH value of the agar after amendment with soluble Seebold (1998) and Seebold, Datnoff, Correa-Victoria & Snyder potassium silicate. (1995) conﬁ rmed this when soluble potassium silicate tested x Means of 10 plates/ treatment. Measurements include as a fungicide resulted in a 40% suppression of rice neck blast. initial 5 mm diam. mycelial disc. Suppression of fungal growth in the current study was shown to y Concentration of the soluble potassium silicate per litre of be dose dependent. However, growth of some fungi such as P. PDA growth medium. perniciosa was actually stimulated at a concentration of 5 ml.ℓ-1 z In each column, values followed by the same letter do not soluble potassium silicate. This phenomenon has been noted in differ signiﬁ cantly (F.Pr < 0.01). numerous fungi with silicon by Wainwright, Al-Wajeeh & Gray-

Table 3. Mean % inhibition of different fungi at different soluble potassium silicate (20.7% SiO2) concentrations and different pH values in ameliorated potato dextrose agar.

SOUTH AFRICAN AVOCADO GROWERS’ ASSOCIATION YEARBOOK 29, 2006 65 Table 4. Mean colony diameter (minus 5 mm diam. initial disc) of different fungi at different soluble potassium silicate concentrations and different pH values in ameliorated PDA on the day when the ﬁ rst concentration reached maximal 80 mm Petri dish diameter.

Figure 1. Mycelial growth of Phytophthora cinnamomi in Figure 2. Mycelial growth of Phomopsis perniciosa in response to 0 ml (pH 5.6), 5 ml (pH 10.3) (B), 10 ml (pH 10.7) response to 0 ml (pH 5.6), 5 ml (pH 10.3) (B), 10 ml (pH 10.7) (C), 20 ml (pH 11.2) (D), 40 ml (pH 11.5) (E), and 80 ml (pH (C), 20 ml (pH 11.2) (D), 40 ml (pH 11.5) (E), and 80 ml (pH 11.7) (F) soluble potassium silicate (20.7% silicon dioxide) 11.7) (F) soluble potassium silicate (20.7% silicon dioxide) per litre of potato dextrose agar (PDA) compared to a per litre of potato dextrose agar (PDA) compared to a potassium hydroxide control group including pH 10.3 (G), potassium hydroxide control group including pH 10.3 (G), pH 10.7 (H), pH 11.2 (I), pH 11.5 (J), and pH 11.7 (K). pH 10.7 (H), pH 11.2 (I), pH 11.5 (J), and pH 11.7 (K). DG H 66 SOUTH AFRICAN AVOCADO GROWERS’ ASSOCIATION YEARBOOK 29, 2006 ston (1997). CARVER, T.L.W., ZEYEN, R.J. & AHLSTRAND, G.G. 1987. The This has implications if soluble silicon is to be used in disease relationship between insoluble silicon and success or failure of attempted control, and it should therefore be ensured that silicon addition primary penetration by powdery mildew (Erysiphe graminis) germlings does not increase ambient silicon concentrations in soil to a level on barley. Physiol. Mol. Plant Pathol., 31: 133-148. conducive to the growth of these fungi. Especially at soluble po- CHERIF, M., ASSELIN, A. & BELANGER, R.R. 1994. Defence responses tassium silicate concentrations between 5 and 20 ml, suppres- induced by soluble silicon in cucumber roots infected by Pythium spp. Phytophathol, 84: 236-242. sion of fungi was variable and at these low concentrations results FAUTEUX, F., RÉMUS-BOREL, W., MENZIES, J.G. & BÉLANGER, R.R. were not consistent between replications. 2005. Silicon and plant disease resistance against pathogenic fungi. Phytophthora cinnamomi was however suppressed even at FEMS Microbiology letters, 249: 1-6. -1 concentrations of 5 ml.ℓ soluble potassium silicate and com- FAWE, A., ABOU-ZAID, M., MENZIES, J.G. & BELANGER, R.R. 1998. pletely inhibited at 10 ml.ℓ-1 and higher concentrations. Based on Silicon-mediated accumulation of fl avanoid phytoalexins in cucumber. the current results and that of previous studies conducted (Kai- Phytopathology, 88: 396-401. ser, Van der Merwe, Bekker & Labuschagne, 2005), a minimum GU, Y.H. & KO, W.-H. 2000. Occurrence of parasexual cycle of dosage of 20 ml per litre of soluble potassium silicate solution Phytophthora parasitica following protoplast fusion. Bot. Bull. Acad. Sin., per litre of water as a soil drench is recommended to ensure 41: 225-230. adequate control of Phytophthora cinnamomi in the fi eld and pot HARDY, G.E. St.J., BARRETT, S. & SHEARER, B.L. 2001. The future trials. of phosphate as a fungicide to control the soilborne plant pathogen The effect of pH on mycelial growth was inconsistent. In all fun- Phytophthora cinnamomi in natural ecosystems. Aust. Plant Pathol., 30: 133-139. gi tested, mycelial growth continued at high pH in the absence of KAISER, C., VAN DER MERWE, R., BEKKER, T.F. & LABUSCHAGNE, soluble potassium silicate, even though at a slower rate. N. 2005. In-vitro inhibition of mycelial growth of several phytopathoge- It can therefore be concluded that soluble potassium silicate nic fungi, including Phytophthora cinnamomi by soluble silicon. South does have a direct fungitoxic activity. Soluble potassium silicate’s African Avocado Growers’ Association Yearbook, 26: 70-74. fungicidal effect was thus the overriding factor especially for fungi KUNOH, H. & ISHIZAKI, H. 1975. Silicon levels near penetration sites such as P. cinnamomi, P. perniciosa and P. maculans (Table 4). of fungi on wheat, barley, cucumber, and morning glory leaves. Physiol. Plant Pathol., 5: 283-287. MENZIES, J., BOWEN, P. & EHRET, D. 1992. Foliar applications of LITERATURE CITED potassium silicate reduce severity of powdery mildew on cucumber, musk- ADATIA, M.H. & BESFORD, R.T. 1986. The effects of silicon on cucumber melon and zucchini squash. J. Amer. Soc. Hort. Sci., 117: 902-905. plants grown in recirculating nutrient solution. Ann. Bot., 58: 343-351. SEEBOLD, K. 1998. The infl uence of silicon fertilization on the ANN, P.J. & KO, W.H. 1992. Survey of antibiotic resistance and development and control of blast, caused by Magnaporthe grisea dependence in Phytophthora. Mycologia, 84: 82-86. (Hebert) Barr, in upland rice. Doctoral dissertation. University of Florida, BEKKER, T.F., KAISER, C., VAN DER MERWE, R. & LABUSCHAGNE, Gainesville, 230 pp. N. 2006. In-Vitro inhibition of mycelial growth of several phytopathogenic SEEBOLD, K., DATNOFF, L., CORREA-VICTORIA, F. & SNYDER, G. fungi by soluble potassium silicate. S. Afr. J. of Plant Soil, 23 (3): 169- 1995. Effects of silicon and fungicides on leaf and neck blast development 172. in rice. Phytopath., 85: 1168. BELANGER, R.R., BOWEN, P.A., EHRET, D.L. & MENZIES, J.G. 1995. WAINWRIGHT, M., AL-WAJEEH, K. & GRAYSTON, S.J. 1997. Effect of Soluble silicon: its role in crop and disease management of greenhouse silicic acid and other silicon compounds on fungal growth in oligotrophic crops. Plant Dis., 79: 329-336. and nutrient rich media. Mycol. Res., 101:933-938. BOWEN, P., MENZIES, J. & EHRET, D. 1992. Soluble silicon sprays ZHENG, X., B. & KO, W.-H. 1997. Continuing variation in colony inhibit powdery mildew development of grape leaves. J. Amer. Soc. Hort. morphology and fungicide sensitivity in Phytophthora cinnamomi Sci., 117: 906-912. following exposure to chloroneb. Bot. Bull. Acad. Sin., 38: 177-182.

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